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, 319 (1-2), 64-78

Protein Transduction as a Means of Effective Manipulation of Cdc42 Activity in Primary T Cells

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Protein Transduction as a Means of Effective Manipulation of Cdc42 Activity in Primary T Cells

Irina Tskvitaria-Fuller et al. J Immunol Methods.

Abstract

The Rho family GTPase Cdc42 is a critical regulator of cellular polarization from yeast to man. An analysis of its function in T cell activation is therefore of interest. This analysis poses two substantial challenges, similar to the analysis of many other critical T cell signaling intermediates. First, Cdc42 is required for development and cell survival, necessitating short-term manipulation of its activity. Second, Cdc42 is likely involved in multiple signaling pathways, requiring approaches to distinguish multiple roles. To address these challenges, we first determined and quantified spatio-temporal patterns of Cdc42 activity using live cell video fluorescence microscopy. This generates hypotheses at which times and locations Cdc42 might play possibly distinct roles. Second and as the focus of this manuscript, we employed protein transduction to manipulate Cdc42 activity for the generation of causality. Protein transduction allows such manipulation to be short-term, quantitative, and with multiple reagents. Here, we characterize uptake, retention, and subcellular distribution of protein transduction reagents. We describe how a more quantitative single cell analysis of Cdc42 activity provides superior distinction between experimental conditions. And we show how we have used dose responses of the protein transduction reagents to minimize side effects while retaining efficacy. We suggest that our strategy is an important complement to more established techniques to study protein function in primary T cells, in particular in the investigation of signaling intermediates that are essential for cell survival and regulate multiple aspects of T cell activation.

Figures

Fig. 1
Fig. 1. tat Cdc42dn uptake and retention in primary T cells
A Fluorescein-conjugated tat Cdc42dn was incubated for 30 min at 37 °C with in vitro primed primary 5C.C7 T cells. Protein uptake and retention were analyzed by FACS with traces displayed on top. As a filled purple trace, autofluorescence of untreated cells is shown. As filled pink and green traces uptake after incubation with 2 or 10 μM tat Cdc42dn is given, respectively. Open traces denote remaining tat Cdc42dn fluorescence after transduction of 5C.C7 T cells with 10 μM tat Cdc42dn and subsequent incubation in the absence of tat Cdc42dn for 1 (dark pink,) 2 (blue), 3 (orange), or 4 h (brown) at 37 °C. On the bottom, mean fluorescence of the traces is plotted as indicated. Data for 0 – 5 h are after initial transduction with 10 μM tat Cdc42dn. Exponential fitting of these data (with a correlation coefficient of R2 = 0.87) was used to determine the half life of tat Cdc42dn. In B, the distribution of expression levels upon retroviral transduction of primary 5C.C7 T cells with actin-GFP is shown for comparison. The large peak on the left is cellular autofluorescence.
Fig. 2
Fig. 2. Subcellular distribution of protein transduction reagents
In A, 5C.C7 T cells were incubated with fluorescein-conjugated tat Cdc42dn or Oregon Green 488, each together with Tetramethylrhodamine-conjugated Transferrin. Representative spinning disk confocal slices of these two-color experiments are shown. Images are scaled comparably throughout. Each column contains images from one cell, as labeled at the right. The Pearson’s coefficient of colocalization (‘Rp’) for the entire cells is given under the images of each cell. In B, a quantitative analysis of the imaging data shown in A is given. 5C.C7 T cells were labeled with TMR-Transferrin together with the reagent listed on the left. Protein transduction reagents were fluorescein-conjugated. Average Pearson’s coefficients with standard deviations are given. An asterisk indicated statistical significance with p < 0.001 against the matching Oregon Green data. 19 – 26 cells (on average 24) were analyzed per experiment. In C, 5C.C7 T cells were incubated with 10 μM tat ZAP-70 tandem SH2-GFP. Matching bright field and fluorescence images (as individual 3D slices) are shown for three T cells. Fluorescence intensity is encoded in a rainbow false color scale (increasing from blue to green, yellow, and red). The uropod is visible as a posterior extension of the T cell. tat ZAP-70 tandem SH2-GFP accumulation at the tip of the uropod can be seen.
Fig. 3
Fig. 3. Inhibition of Cdc42 activation at the T cell/APC interface without concomitant blockade of cell couple formation is saturated at 100 nM tat Cdc42dn and 1 μM tat WASP GBD
Cdc42 biosensor-transduced 5C.C7 T cells were activated by CH27 APCs under the indicated conditions. The agonist peptide concentration was 10 μM MCC (full stimulus) unless noted otherwise. ‘anti-B7’ indicates 10 μg/ml anti-B7-1 plus anti-B7-2, ‘limiting’ indicates 0.1 μM MCC plus anti-B7 plus 10 μg/ml anti-ICAM-1 antibody. Accumulation (A) and centrality (B) indices are given within the first (1’) and second (2’) minute after cell couple formation. For the 1 min time point, data from 0:00/0:20/0:40 after tight cell couple formation were pooled, for the 2 min time point, data from 1:00/1:20/1:40. Within the first minute distinct and dramatic morphologically changes occur. In the subsequent about four minutes, represented here by the 2’ data, proximal T cell signaling peaks. The concentrations of tat Cdc42dn and tat WASP GBD added are listed. ‘100/350 nM Cdc42dn’ and ‘1/3 μM WASP GBD’ refer to pooled data for 100 nM and 350 nM tat Cdc42dn and for 1 μM and 3 μM tat WASP GBD, respectively. Data for the presence of 100 nM tat Rac1dn are shown to allow assessment of the overlap in Cdc42dn and Rac1dn function. p-values (Student’s t-test) versus 10 μM MCC are given at the right of each panel in parentheses. The left number refers to the 1 min data, the right number to the 2 min data. ‘n.s.’ indicates p > 0.05. In C, representative images of Cdc42 biosensor-transduced 5C.C7 T cells are given. T cells were activated with a CH27 B cell lymphoma APC in the presence of 10 μM MCC agonist peptide (full stimulus). Top down maximum projections of 3-dimensional Cdc42 sensor fluorescence data are shown. The Cdc42 sensor fluorescence intensity is displayed in a rainbow false color scale (increasing from blue to green, yellow, and red). APCs are not visible. In the four panels from left to right, the T cell/APC interfaces are on the bottom right, bottom left, right, and bottom, respectively. The fraction of sensor translocated to the interface (‘% accumulation’) and the ratio of (the fraction of sensor translocated to the interface) to (the area of accumulation as a fraction of the interface area) (‘% acc/area’), i.e. the two single cell measures that are part of the accumulation and centrality indices are given under each cell. The first and fourth image from the left represent high/low accumulation, the second and third high/low centrality.
Fig. 4
Fig. 4. Inhibition of Cdc42 activation at the T cell/APC interface without concomitant blockade of cell couple formation is saturated at 100 nM tat Cdc42dn and 1 μM tat WASP GBD
An alternate analysis of the data presented in Fig. 3 is shown. % cell couples with sensor accumulation in any pattern (A) or in a central pattern (B) are given within the first (1’) and second (2’) minute after cell couple formation as in Fig. 3. ‘% cell couples with accumulation in any pattern’ is the same number as the ‘fraction of cell couples above threshold’ in the calculation of the accumulation and centrality indices.
Fig. 5
Fig. 5. Limiting T cell activation conditions interfere with T cell couple formation
The percentage of primary 5C.C7 T cells contacting a CH27 APC under the indicated conditions (top four bars as Fig. 3; bottom four bars 10 μM MCC with increasing concentrations of tat Cdc42dn) that form a tight cell couple is given with standard deviations. One/two asterisks indicate statistical significance against the matching 10 μM MCC data set with p ≤ 0.05/0.001, respectively (Student’s t-test). For the top four conditions, on average 242 (205 – 294) T cells were analyzed per condition from at least two independent groups of experiments. The bottom four conditions are a duplicate of the data presented in (Tskvitaria-Fuller et al., 2006) to facilitate comparison. On average 22 cells from at least two independent experiments were analyzed.
Fig. 6
Fig. 6. Cdc42dn and Rac1dn block Cdc42 activity to the same extent when analyzed biochemically
Cdc42 GTP was precipitated from 5C.C7 T cell extracts with the PAK GBD and quantified by Western blotting. Data are given as the average amount of precipitated Cdc42 GTP ± standard deviation relative to a full stimulus. The T cell activation conditions were: 10 μg/ml anti-TCR plus 10 μg/ml anti-CD28 (full stimulus), 10 μg/ml anti-TCR only, 0.1 μg/ml anti-TCR plus 10 μg/ml anti-CD28 (low anti-TCR), and 10 μg/ml anti-TCR plus 10 μg/ml anti-CD28 plus 100 nM tat Cdc42dn or 100 nM tat Rac1dn. 2 - 4 independent experiments were performed per condition.

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